The ribosome is one of the best antibiotic targets in the cell. The majority of antibiotics that act upon the ribosome binds to the ribosomal RNA and inhibits protein synthesis by interfering with rRNA functions. The great advantage of rRNA as an antibiotic target is that rRNA is encoded in multiple gene copies. Because of that, mutations in at least several gene copies are required to render cells drug resistant through alteration of the antibiotic-target site. This delays the appearance of natural resistant isolates and complicates deliberate engineering of resistant mutants. The goal of the proposal is to identify sites in rRNA that can be used as antibiotic targets and to develop new antibiotics that inhibit B. anthracis protein synthesis by interacting with these sites. The proposal is comprised of four major steps. Firstly, we will identify prospective antibiotic sites in ribosomal RNA. B y mapping lethal mutations in rRNA we will pinpoint the ribosomal sites where small alterations in the rRNA structure cause severe defects in protein synthesis. The other rRNA target sites will be chosen through rational approach. At the second step, the selected rRNA sites will be validated as antibiotic targets. The rRNA structures of the B. anthracis ribosome will be used to select short peptides capable of binding to these sites. Such peptides will be then chemically synthesized and tested in vitro in binding and protein synthesis inhibition experiments. The atomic structure of the RNA-peptide complexes, the mode of the peptide binding and the mechanism of inhibition of protein synthesis will be determined. At the third step, the validated target rRNA sites will be used for the identification of chemical lead compounds that can inhibit B. anthracis protein synthesis due to the interaction with these sites in the ribosome. The lead compounds will be identified using rational design and docking algorithms in conjunction with high-throughput screening of libraries of chemical compounds. The lead compounds will be optimized and tested in in vitro and in vivo models. Finally, at the fourth step of the proposed research, the detailed structure of the complex of the antibiotic compounds with their target site in the ribosome will be investigated, the mechanism of inhibition of protein synthesis will be studied and possible resistance mechanisms will be explored. The obtained information will be used for further optimization of the antibiotic leads.